Polysaccharide containing phosphorylcholine group and process for producing the same

ABSTRACT

The present invention is a method for manufacturing a phosphorylcholine group-containing polysaccharide wherein the aldehyde derivative-containing compound obtained by the oxidative ring-opening reaction of glycerophosphorylcholine is added to a polysaccharide containing amino groups as well as a new polysaccharide having phosphorylcholine groups obtained from this manufacturing method. 
     The object of the present invention is to provide a phosphorylcholine group-containing polysaccharide that is superior in biocompatibility and moisture retention, and is useful as a polymer material for medical use, as well as a simple method of manufacturing it. 
     The polysaccharide of the present invention is utilized, for example, in artificial organs, biomembranes, coating agents for medical tools, drug delivery, and in cosmetics.

TECHNICAL FIELD

The present invention relates to polysaccharides containingphosphorylcholine groups and methods for manufacturing them.

The phosphorylcholine group-containing polysaccharide of the presentinvention is superior in biocompatibility and moisture retention, and isuseful as a polymer material for medical use. Specifically, it isutilized in artificial organs, biomembranes, coating agents for medicaltools, drug delivery, and in cosmetics.

BACKGROUND ART

Macromolecules containing phosphorylcholine groups have been developedas biocompatible materials. Polymers having phosphorylcholine groupshave been synthesized mainly as follows: acryl type monomers mainlyhaving hydroxyl groups and 2-chloro-1,3,2-dioxaphosphorane-2-oxide arebrought into reaction and then trimethylamine is used to turn thereaction product into quaternary ammonium to synthesize monomers havinga phosphorylcholine structure, which are then polymerized.

However, due to the monomer solubility issues when introducing thehydrophobic groups, this method requires the use of an organic solventknown as a chain transfer catalyst such as methanol, ethanol, andchloroform as a polymerization solvent, which makes it difficult toproduce high molecular weight polymers. Also, the monomer synthesisreaction has to be conducted under strictly anhydrous conditions, whichcomplicates the technique.

In addition the conventional manufacturing method that polymerizesmonomers having phosphorylcholine on side chains has a problem in thatthe steric hindrance of the phosphorylcholine group reduces thepolymerization yield or makes it impossible to obtain the desiredpolymer.

In view of the description above, the inventors conducted earnestresearch on the manufacturing method of the phosphorylcholinegroup-containing polymer, and completed the present invention bydiscovering that polysaccharides having the phosphorylcholine structurecan be obtained easily and with a high versatility by reacting acompound containing phosphorylcholine groups with a polysaccharidehaving a functional group that reacts with this compound, which leads toa macromolecular reaction in the side chains of the polymer.

DISCLOSURE OF INVENTION

That is, the present invention provides a polysaccharide having aphosphorylcholine group represented by the following general formula(1).

That is, the present invention provides a polysaccharide having aphosphorylcholine group represented by the following general formulas(2)-(10).

In the general formulas (2)-(7) n denotes an integer 1-22, m denotes aninteger 1-20, and SUGAR denotes a polysaccharide.

In general formulas (8)-(10), R1, R2, and R5 denote O, NH, or a tertiaryamine.

R3 and R4 are straight chain or branched alkylene having 1-22 carbonatoms, or ethylene oxide having 1-20 repeat units.

R6 denotes a hydrocarbon including aromatic hydrocarbons or aperfluoroalkylene group having 1-22 carbon atoms.

k denotes an integer 0-6, n, m, and q denote positive integers, and“sugar” denotes a polysaccharide.

Furthermore, the present invention provides a method for manufacturing apolysaccharide having phosphorylcholine groups wherein the aldehydederivative-containing compound obtained by the oxidative ring-openingreaction of glycerophosphorylcholine is added to a polysaccharidecontaining amino groups.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a scheme for preparing a monofunctional aldehyde derivativecontaining a phosphorylcholine group.

FIG. 2 shows a preparation scheme for the polysaccharide represented bygeneral formula (2).

FIG. 3 shows a structural formula and NMR spectrum of synthesis example1.

FIG. 4 is a structural formula of synthesis example 2.

FIG. 5 shows a structural formula and NMR spectrum of synthesis example3.

FIG. 6 shows a structural formula and NMR spectrum of synthesis example4.

FIG. 7 is a structural formula of synthesis example 5.

FIG. 8 is a structural formula of synthesis example 6.

FIG. 9 is a structural formula of synthesis example 7.

FIG. 10 shows a structural formula and NMR spectrum of synthesis example8.

FIG. 11 is a structural formula of synthesis example 9.

FIG. 12 shows a structural formula and NMR spectrum of synthesis example10.

FIG. 13 is a structural formula of synthesis example 11.

FIG. 14 is a structural formula of synthesis example 12.

FIG. 15 is a structural formula of synthesis example 13.

FIG. 16 is a structural formula of synthesis example 14.

FIG. 17 is a structural formula of synthesis example 15.

FIG. 18 is a structural formula of synthesis example 16.

FIG. 19 is a structural formula of synthesis example 17.

FIG. 20 is a structural formula of synthesis example 18.

FIG. 21 is a structural formula of synthesis example 19.

FIG. 22 is a structural formula of synthesis example 20.

FIG. 23 is a structural formula of synthesis example 21.

FIG. 24 is a graph showing the hemolysis test results.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below.

The preparation method of the polysaccharide containingphosphorylcholine groups of the present invention is as follows.

[1]: A polysaccharide having amino groups is brought into a reductiveamination reaction with a hydrate derivative or aldehyde derivativeobtained by the oxidative ring-opening reaction ofglycerophosphorylcholine to obtain a polysaccharide to whichphosphorylcholine groups are added.

A polysaccharide having phosphorylcholine groups added to its main chainhas not been reported. The only method known for such a polysaccharideuses graft polymerization to indirectly introduce phosphorylcholinegroups to side chains away from the main chain (Journal of BiomedicalMaterials Research, Vol. 29, 181-188 (1995)), but this method has theshortcoming of being cumbersome.

The preparation method of the present invention using the reactiondescribed in [1] has significant advantages in that the introductionyield is high and the introduction ratio can be controlled easily.

For example, the introduction ratio of phosphorylcholine can becontrolled to change the hydrophilicity of the polymer or to adapt torequired biocompatibility. Also, free from the influence of thephosphorylcholine groups, sugar chains can be given required functionsby means of hydrophobic groups and such, and then any quantity ofphosphorylcholine groups can be added to easily obtain the targetfunctional polymer material. A form of polymer that introducesphosphorylcholine in the main chain of a polysaccharide, or a form ofpolymer that introduces phosphorylcholine into the main chain of thepolymer introduced to a polysaccharide can be synthesized, allowingflexibility according to the application.

In the preparation method [1] in the present invention, the compoundcontaining the aldehyde derivative obtained by the oxidativering-opening reaction of glycerophosphorylcholine is obtained byoxidative ring-opening of the prior art glycerophosphorylcholine groupby means of a prior art method, which is a very easy step.

This reaction uses periodic acid or periodate to oxidize 1,2-diol toopen the bond and obtain two aldehyde derivatives; in this particularmethod, a phosphorylcholine aldehyde derivative and formaldehyde areproduced. The reaction is usually carried out in water or in an organicsolvent containing water. The reaction temperature is between 0° C. toroom temperature. The aldehyde derivative may go through the equilibriumreaction in water to become a hydrate, but this does not affect thesubsequent reaction with the amine.

Selection of the polysaccharide having amino groups is not limited inparticular. It suffices if the side chains of the polysaccharide haveamino groups with which the aldehyde derivative obtained by theoxidative ring-opening reaction of glycerophosphorylcholine can react.

Prior art polysaccharides can be used. A prior art method can be used tointroduce amino groups into a prior art polysaccharide to obtain apolysaccharide tailored for a target application.

Examples of the polysaccharide include dextran, cellulose, hyaluronicacid, pullulan, glucomannan, chondroitin sulfate, agarose, pectin,chitin, chitosan, gum Arabic, carrageenan, gellan, guar gum, alginicacid, xanthan gum, amylose, and rheozan.

Amino groups can be added to the polysaccharide by, for example,introduction of carboxylic acid via the carboxymethylation reactionfollowed by the amidation reaction with diamine. A polysaccharidecontaining amino groups, such as chitosan, can be used for thephosphorylcholine introduction reaction without further treatment. Thephosphorylcholine group content of the final target product can bedesigned by controlling the amino group content.

Amino group-containing polysaccharides can also be obtained by reductiveamination coupling between common polysaccharides having reductiveterminals and polymers having amino groups such as polylysine orpolyethyleneimine.

The reductive amination reaction for bonding the aldehyde derivative (orhydrate derivative polymer) obtained by the oxidative ring-openingreaction of glycerophosphorylcholine to the amino groups of the polymercan be carried out easily by stirring both of them in a solvent.

This reaction is carried out by dissolving those two in water or alcohol(a third organic solvent ingredient can be mixed in, too) to form animine and reducing it with a reducing agent to obtain a secondary amine.

For the reducing agent, a mild reducing agent such as sodiumcyanoboronate is preferable, but other reducing agents can be used aslong as the phosphorylcholine is stable. The reaction is usually carriedout at 0° C. to room temperature, but heating may be done depending onthe situation.

Using the aforementioned preparation method, a polysaccharide containinga desired amount of phosphorylcholine groups in the hydrophilic portionis easily obtained.

It is also possible to design a biocompatible polymer having thestructure of biomembrane components such as phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and diphosphatidylglycerol.

The hydrophilic portion of the polysaccharide may include a carboxylategroup, hydroxyl group, primary-tertiary amine group, sulfonate group,phosphate group, polyoxyethylene group, ammonium group, amide,carboxybetaine, and saccharide, and the type and content of these in thepolysaccharide can be adjusted to design its functions.

As for the hydrophobic portion of the polysaccharide, depending on theapplication, straight chain or branched alkyls having 1-22 carbon atoms,cyclic alkyls such as cholesterol, alkyl groups containing unsaturatedbonds such as oleyl, hydrocarbon type aromatics such as benzene rings,naphthalene rings, and pyrene, hetero type aromatics such as pyridinerings, imidazole, thiazole, and indole, and hydrophobic groups such asperfluoroalkyl and polyalkylsiloxane can be introduced for moleculardesign.

The hydrophobic group can bond directly to the main chain with theester, ether, amide, urethane, or urea bond, or indirectly via a spacer.Examples of the spacer include hydrophilic polyethyleneoxide,hydrophobic polypropyleneoxide, and straight chain alkyls having 2-22carbon atoms.

Using the aforementioned preparation method, the polysaccharides of thepresent invention represented in general formulas (1)-(10) can easily beprepared.

The polysaccharides represented by general formulas (2)-(10) have thecharacteristic of having a polysaccharide and phosphorylcholine bondedvia a secondary amine. Phosphorylcholine directly bonds to the polymermain chain via a secondary amine.

The preparation method of the present invention can be used to bond apolysaccharide and phosphorylcholine via a secondary amine and also tobond phosphorylcholine in the polymer prepared by bonding an acrylicpolymer and such to sugar terminals in the block fashion.

The polysaccharide having phosphorylcholine groups of the presentinvention is a polysaccharide polymer material with superiorhydrophilicity and moisture retention.

In general formulas (2)-(10), “sugar”, which represents apolysaccharide, can contain hetero-aromatic groups, aromatic groups,perfluoroalkyl groups, and straight chain or branched alkyl groupshaving 1-22 carbon atoms.

The polysaccharides of general formulas (8)-(10) are polymers composedof phosphorylcholine groups and polysaccharides added to polymer mainchains.

Such a polymer is the result of the addition of polysaccharide andphosphorylcholine groups via amino groups; it is a polymer containingtwo or three types of repeat units represented by parentheses followedby m, n, or q; usually, the repeat units to which the polysaccharide andphosphorylcholine groups are added are randomly polymerized. m, n, and qare positive integers; they indicate the composition of the polymer interms of the repeat units to which the polysaccharide andphosphorylcholine groups, respectively, are added.

Since preparation is done by the polymer reaction in which thepolysaccharide and the phosphorylcholine groups are added to a polymerhaving amino groups, the polymer can contain repeat units in which aminogroups remain to which no polysaccharide or phosphorylcholine groups areadded.

Selection of the polysaccharide in general formulas (2)-(7) is notlimited in particular; any polysaccharide having hydroxyl groups andsoluble in the reaction solvent can be used.

Selection of the polysaccharide in general formulas (8)-(10) is notlimited in particular; any polysaccharide having reductive terminals andsoluble in the reaction solvent can be used.

The polysaccharides having phosphorylcholine groups in general formulas(2)-(10) can be easily prepared from the polysaccharides containingamino groups represented by the following general formulas (11)-(19) bymeans of the preparation method of the present invention. Other thanthese general formulas, sugar that naturally has amino groups (such aschitosan) can be used.

In the general formulas (11)-(16) n denotes an integer 1-22, m denotesan integer 1-20, and SUGAR denotes a polysaccharide.

In general formulas (17)-(19), R1, R2, and R5 denote O, NH, or atertiary amine.

R3 and R4 are straight chain or branched alkylenes having 1-22 carbonatoms, or ethylene oxide having 1-20 repeat units.

R6 denotes a hydrocarbon including aromatic hydrocarbons or aperfluoroalkylene group having 1-22 carbon atoms.

k denotes an integer 0-6, n, m, and q denote positive integers, and“sugar” denotes a polysaccharide.

FIG. 1 shows a scheme for preparing a monofunctional aldehyde derivativecontaining a phosphorylcholine group, and FIG. 2 shows a preparationscheme for the polysaccharide represented by general formula (2).

These show that the target phosphorylcholine group-containingpolysaccharide of the present invention can be easily obtained from themonofunctional phosphorylcholine aldehyde derivative by using themanufacturing method of the present invention.

The polysaccharides of general formulas (3)-(11) can also be obtained inthe same manner.

EXAMPLES

Specific synthesis examples follow. The present invention is not limitedto the following synthesis examples.

The composition of the polysaccharides of the present invention can bedetermined by NMR.

Synthesis Example 1 An aldehyde derivative containing aphosphorylcholine group

L-α-glycerophosphorylcholine (450 mg) is dissolved in 15 ml of distilledwater and cooled in an ice water bath. Sodium periodate (750 mg) isadded and two hours of stirring is carried out. Furthermore, ethyleneglycol (150 mg) is added and overnightstirring is carried out. Thereaction solution is vacuum-concentrated and vacuum-dried and the targetsubstance is extracted with methanol.

The structural formula and the NMR spectrum are shown in FIG. 3.

Synthesis Example 2 Synthesis of carboxymethyldextran

Dextran (5 g) and chloroacetic acid (10.28 g) are dissolved in a 6Nsolution of sodium hydrochloride, followed by heating and stirring forone hour at 60° C. After cooling the mixture down to room temperature,the target substance is obtained by means of reprecipitation inmethanol. (Yield 6.2 g).

The structural formula is shown in FIG. 4.

Synthesis Example 3 Synthesis of aminodextran

The carboxydextran (1 g) of Synthesis example 2 and ethyelenediamine (10ml) are dissolved in distilled water (10 ml) and the pH is adjusted tofive. 1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5g) is gradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water and 1.25 g of the targetsubstance is obtained by means of lyophilization.

The structural formula and the NMR spectrum are shown in FIG. 5.

Synthesis Example 4 Synthesis of aminocellulose

Carboxymethylcellulose (1 g) and ethyelenediamine (10 ml) are dissolvedin distilled water (10 ml) and the pH is adjusted to five.1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5 g) isgradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water and 1.05 g of the targetsubstance is obtained by means of lyophilization.

The structural formula and the NMR spectrum are shown in FIG. 6.

Synthesis Example 5 Synthesis of aminohyaluronic acid

Hyaluronic acid (1 g) and ethyelenediamine (10 ml) are dissolved indistilled water (10 ml) and the pH is adjusted to five.

1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5 g) isgradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water and 1.2 g of the target substanceis obtained by means of lyophilization.

The structural formula is shown in FIG. 7.

Synthesis Example 6 Synthesis of carboxymethylpullulan

Pullulan (5 g) and chloroacetic acid (10.28 g) are dissolved in a 6Nsodium hydrochloride solution, followed by one hour of heating andstirring at 60° C. After cooling the mixture down to room temperature,the target substance is obtained by means of reprecipitation inmethanol. (Yield 5.1 g).

The structural formula is shown in FIG. 8.

Synthesis Example 7 Synthesis of aminopullulan

The carboxypullulan (1 g) of Synthesis example 6 and ethyelenediamine(10 ml) are dissolved in distilled water (10 ml) and the pH is adjustedto five. 1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride(1.5 g) is gradually added. After stirring overnight at roomtemperature, the reaction solution is dialyzed in water and 1.15 g ofthe target substance is obtained by means of lyophilization.

The structural formula is shown in FIG. 9.

Synthesis Example 8 Synthesis of phosphorylcholinedextran

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe aminodextran (1 g) solution (15 ml) of Synthesis example 3, followedby stirring for five hours at room temperature. Sodium cyanoboratehydride (500 mg) is added, followed by overnight stirring. The targetsubstance (1.1 g) is obtained after purification by means of dialyzationand lyophilization.

The structural formula and the NMR spectrum are shown in FIG. 10.

Synthesis Example 9 Synthesis of phosphorylcholinecellulose

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe aminocellulose (1 g) solution (15 ml) of Synthesis example 4,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.05 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 11.

Synthesis Example Synthesis of phosphorylcholinehyaluronic acid

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe aminohyaluronic acid (1 g) solution (15 ml) of Synthesis example 6,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.2 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula and the NMR spectrum are shown in FIG. 12.

Synthesis Example Synthesis of phosphorylcholinepullulan

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe aminopullulan (1 g) solution (15 ml) of Synthesis example 8,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (0.99 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 13.

Synthesis Example Synthesis of hydrophobicized phosphorylcholinedextran

A DMF solution (15 ml) of lauric acid (0.02 g) is added to theaminodextran (1 g) solution (15 ml) of Synthesis example 3, and1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5 g) isgradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water, and the phosphorylcholinealdehyde (1 g) of Synthesis example 1 is added to this aqueous solution,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.1 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 14.

Synthesis Example 13 Synthesis of hydrophobicizedphosphorylcholinecellulose

A DMF solution (15 ml) of stearic acid (0.01 g) is added to theaminodextran (1 g) solution (15 ml) of Synthesis example 4, and1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5 g) isgradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water, and the phosphorylcholinealdehyde (1 g) of Synthesis example 1 is added to this aqueous solution,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (0.89 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 15.

Synthesis Example Synthesis of hydrophobicizedphosphorylcholinehyaluronic acid

A DMF solution (15 ml) of perfluorooctanoic acid (0.2 g) is added to theaminohyaluronic acid (1 g) aqueous solution (15 ml) of Synthesis example5, and 1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5g) is gradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water, and the phosphorylcholinealdehyde (1 g) of Synthesis example 1 is added to this aqueous solution,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.2 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 16.

Synthesis Example 15 Synthesis of hydrophobicizedphosphorylcholinepllulan

A DMF solution (15 ml) of lauric acid (0.02 g) is added to theaminopullulan (1 g) aqueous solution (15 ml) of Synthesis example 7, and1{3-(dimethylamino)propyl}3-ethylcarbodiimide hydrochloride (1.5 g) isgradually added. After stirring overnight at room temperature, thereaction solution is dialyzed in water, and the phosphorylcholinealdehyde (1 g) of Synthesis example 1 is added to this aqueous solution,followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.1 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 17.

Synthesis Example 16 Synthesis of hyaluronic acid-polylysine

Hyaluronic acid (1 g) and polylysine (1 g) are dissolved in distilledwater (15 ml) and stirred at room temperature for five hours. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.85 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 18.

Synthesis Example Synthesis of dextran-polyallylamine

Dextran (1 g) and polyallylamine (1 g) are dissolved in distilled water(15 ml) and stirred at room temperature for five hours. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.6 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 19.

Synthesis Example 18 Synthesis of hydroxyethylcellulose-polyN-isopropylacrylamide-poly

N-(3-aminopropyl)methacrylamide Hydroxyethylcellulose (1 g) andhydroxyethylcellulose-poly N-isopropylacrylamide-polyN-(3-aminopropyl)methacrylamide 1:1 copolymer (1 g) are dissolved indistilled water (15 ml) and stirred at room temperature for five hours.Sodium cyanoborate hydride (500 mg) is added, followed by overnightstirring. The target substance (1.5 g) is obtained after purification bymeans of dialyzation and lyophilization.

The structural formula is shown in FIG. 20.

Synthesis Example 19 Synthesis of Hyaluronicacid-phosphorylcholinepolylysine

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe hyaluronic acid-polylysine (1 g) aqueous solution (15 ml) ofSynthesis example 17, followed by stirring for five hours at roomtemperature. Sodium cyanoborate hydride (500 mg) is added, followed byovernight stirring. The target substance (1.0 g) is obtained afterpurification by means of dialyzation and lyophilization.

The structural formula is shown in FIG. 21.

Synthesis Example 20 Synthesis ofdextran-phosphorylcholinepolyallylamine

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe polyallylamine (1 g) aqueous solution (15 ml) of Synthesis example18, followed by stirring for five hours at room temperature. Sodiumcyanoborate hydride (500 mg) is added, followed by overnight stirring.The target substance (1.2 g) is obtained after purification by means ofdialyzation and lyophilization.

The structural formula is shown in FIG. 22.

Synthesis Example 21 Synthesis ofhydroxyethylcellulose-phosphorylcholine poly N-isopropylacrylamide-polyN-(3-aminopropyl)methacrylamide

The phosphorylcholine aldehyde (1 g) of Synthesis example 1 is added tothe hydroxyethylcellulose-poly N-isopropylacrylamide-polyN-(3-aminopropyl)methacrylamide (1 g) aqueous solution (15 ml) ofSynthesis example 19, followed by stirring for five hours at roomtemperature. Sodium cyanoborate hydride (500 mg) is added, followed byovernight stirring. The target substance (0.98 g) is obtained afterpurification by means of dialyzation and lyophilization.

The structural formula is shown in FIG. 23.

The polysaccharides of the present invention (Synthesis examples 8-15)synthesized as described above were used for human blood hemolysis testsconducted with the following procedure.

“Hemolysis Test”

Human blood is added to a K3 solution containing EDTA (5.5 mg), followedby centrifugation at 200 G for five minutes at 4° C. The obtained bloodcells are rinsed three times with phosphate buffer (PBS), and mixed witha PBS solution of the polymer. After a 20-minute incubation at 37° C.,centrifugation at 5,300 G for five minutes at 4° C. was conducted. Thedegree of hemolysis (%) was evaluated from UV absorption (541 nm) of thesupernatant.

The degree of hemolysis (%) is determined by the following equation.Degree of hemolysis (%)={(UV absorption of the supernatant of the bloodto which the polymer is added)−(UV absorption of the supernatant of theblood without the added polymer)}/{(UV absorption of the supernatant ofthe completely hemolyzed blood)−(UV absorption of the supernatant of theblood without the added polymer)}×100

The results of the hemolysis test and the degree of hemolysis (%), areshown in Table 1 and FIG. 24. In the graph in FIG. 24, the degree ofhemolysis (%) for the polymers to which the phosphorylcholine groupsprepared in Synthesis examples 8-15 are introduced is in every caseapproximately 0%, overlapping with the horizontal axis, and no hemolysisreaction is indicated. This indicates that all of the polysaccharides(polymers) to which phosphorylcholine groups are introduced according tothe present invention have a very high blood compatibility.

TABLE 1 “Hemolysis test results” Polymer Concentration SynthesisSynthesis Synthesis Synthesis Synthesis Synthesis Synthesis SynthesisAcrylic (g/L) example 8 example 9 example 10 example 11 example 12example 13 example 14 example 15 acid 0 0 0 0 0 0 0 0 0 0 0.1 0.01 00.01 0.01 0 0 0.02 0 10 0.25 0.02 0 0.01 0.01 0 0 0.03 0 15 0.5 0.03 00.05 0.01 0 0 0.035 0 35 1 0.02 0 0.1 0.03 0 0 0.04 0.1 50 2.5 0.02 0.050.1 0.03 0 0 0.04 0.1 100 5 0.025 0.1 0.1 0.04 0.11 0.16 0.05 0.15 10010 0.05 0.1 0.1 0.05 0.4 0.11 0.07 0.2 100

INDUSTRIAL APPLICABILITY

The phosphorylcholine group-containing polysaccharide of the presentinvention has high biocompatibility and moisture retention and is auseful polymer material; it has a variety of applications such asartificial organs, biomembranes, coating agents for medical tools, drugdelivery, and as cosmetic ingredients.

The manufacturing method of the present invention has a great advantagein that it allows flexible designing of phosphorylcholinegroup-containing polymers ideal for biocompatible polymer materials.

For example, initially the most preferable material for the applicationcan be obtained through a designing process unrestricted by the presenceof phosphorylcholine groups by introducing hydrophobic groups into thepolysaccharide, for example. Subsequently, a desired quantity ofphosphorylcholine groups can be easily added to obtain the targetfunctional polymer material.

1. A polysaccharide having a phosphorylcholine group represented by oneof the following general formulas (2)-(10):

wherein, in general formulas (2)-(7), n denotes an integer 1-22; ingeneral formulas 3 and 4 m denotes an integer 1-22; in general formulas(2)-(7), SUGAR denotes a polysaccharide; in general (10), R1, R2, and R5denote O, NH, or a tertiary amine; R3 and R4 are straight chain orbranched alkylenes having 1-22 carbon atoms, or ethylene oxide having1-20 repeat units; R6 denotes a hydrocarbon including aromatichydrocarbons or a perfluoroalkylene group having 1-22 carbon atoms; Kdenotes an integer 0-6; in formulas (8),(9) and (10), n, m and q denotepositive integers, and “sugar” denotes a polysachharide.